The use of genetically encoded fluorescent activity probes represent the most advanced method to monitor the electrical activity of networks of neurons without using electrodes. While genetically encoded calcium indicators have been evolved to produce robust signals in a variety of different neuronal preparations, fluorescent probes of membrane potential have not been well evolved. Current voltage probes, while finally in expanded use, will need considerable improvement if the goal of recording the activity of a large number of neurons simultaneously in vivo is to be achieved. The goal of this project is to discover protein-based fluorescent voltage probes with signal to noise characteristics that allow routine optical recording of action potentials from single cortical neurons in vivo. We are seeking probes with significantly improved signal to noise characteristics, red-shifted fluorescence spectra, faster on and off rates and better plasma membrane expression. This project brings together leading genetic probe scientists to: design new probe scaffolds, robotically screen large incrementally modified libraries of probes, identify probes with improved response properties, then validate these new probes under standardized experimental conditions in a range of 'real world' neuronal preparations with increasing levels of complexity. Finally, we will make all probe reagents (i.e. plasmid DNA, AAV particles, transgenic flies, etc.) as well as supporting validation data readily available to the research community.